The synthesis of DNA by chemical methods has led to the production of a variety of synthetic genes, e.g. human leukocyte interferon gene [Edge, M. D. et al.; Nature 292, 756 (1981)]. If synthetic genes are recombined with expression vectors and transferred into the appropriate microorganism or eucaryotic cell, a genotypically and phenotypically mutant organism can be produced that can correctly transcribe and translate the artifical gene.
Applicants have synthesized and cloned a sequence coding for human epidermal growth factor (hereinafter hEGF). Applicants have employed stepwise solid-phase synthesis of oligonucleotides of single strands, followed by annealing and ligation. The codon usage in the sequence is designed for maximal expression in yeast. This invention also provides inter alia processes for cloning and amplification of the sequence coding for hEGF, methods for purifying it, and methods for constructing DNA expression vectors carrying hEGF.
Epidermal Growth Factor (EGF) has a broad spectrum of biochemical and physiological effects on cells, including, e.g., accelerated proliferation or differentiation, potentiation of limited forms of carcinogenesis, stimulation of transport, activation of glycolysis, stimulation of macromolecular synthesis, and other activities unrelated to mitogenesis, such as inhibition of gastric secretion. A useful discussion of these functions as well as review of the structure and amino acid sequence of EGF may be found in Carpenter, G. et al. Ann. Rev. Biochem., 48, 193 (1979). See also Gospodarowicz, D. Ann. Rev. Physiol. 43, 251 (1981).
Of the epidermal growth factors that have been purified to date, mouse epidermal growth factor is the best characterized as to its physical, chemical and biological properties. Mouse EGF is an acidic protein of about 53 amino acids with three disulfide bonds. In interacting with the cell, it is probable that EGF binds to a cell receptor, yielding a complex which is internalized, then degraded by lysosomes. One intriguing property is its capacity to stimulate intracellular phosphorylation of tyrosine residues, a known activity of src proteins.
Processes and methods for synthesizing DNA sequences coding for urogastrone, a polypeptide related to EGF, were published in EPO application 46,039, filed on or about July 31, 1981, by applicant G. D. Searle and Co. The Searle sequence and its corresponding protein are substantially different from the present invention for the following reasons. The DNA sequence of the Searle reference codes for a procaryote prepeptide of about an additional 14 amino acids on the amino terminus of urogastrone, an added sequence required for adequate and effective secretion in a procaryotic host. One embodiment of the present invention dispenses with a prepeptide sequence and employs a eucaryote host for propagation and expression of EGF.
Other differences between the Searle application and the present application are, secondly, that the commercial use of procaryotic cells as hosts for synthesizing human proteins requires scrupulous and expensive purification procedures to avoid the effects of pyrogens and other endotoxins. The yeast cells used in one embodiment of the present invention provide extracts entirely or substantially free of pyrogenic material, hence contaminants do not provide a hazard to the public. Yeast also provides good fermentation volume.
Thirdly, the DNA sequence of the present invention has the codon bias of yeast cells, allowing for highly efficient expression of the DNA sequence in an appropriate host cell. In addition, applicants have demonstrated expression of EGF whereas those of the Searle application have not.
Finally, the present applicants provide modifications in the techniques of solid-phase DNA synthesis that allow the synthesis of much larger blocks of single-stranded oligodeoxyribonucleotides than has previously been practical. These larger blocks reduce the number of annealing and ligation steps, a result that substantially increases the yield of final product.
The protein hEGF is useful for modulating the growth and culture of mammalian cells, particularly human cells, on a commercial scale. Potential uses include clinical treatment of wounds and ulcers.